Cerebrospinal fluid (CSF) is present between two meningeal membranes of the brain and is circulated over the cerebral hemispheres and spinal cord. The CSF acts as a protective cushion for the underlying central nervous tissue; other functions include collection of wastes, circulation of nutrients, and lubrication of the central nervous system.
The major clinical role of CSF analysis is in the diagnosis of bacterial meningitis, the differential diagnosis of viral and fungal meningitis, encephalitis, neurological disorders and the diagnosis of leukemias with CSF involvement. Other indications for CSF analysis include the monitoring of patients undergoing therapy for leukemias and lymphomas. The examination of CSF samples typically involves chemical and immunological studies, and, more particularly, microbiologic examination and hematological analysis to derive red blood cell count (RBC), white blood cell count (WBC) and WBC differential cell count. These results are correlated with clinical findings and radiographic studies to provide a clinical diagnosis.
Hematological analysis of CSF specimens is currently performed in the majority of hospital laboratories using manual cell counting and cell differentiation methods. These analyses are among the most laborious manual procedures in the clinical laboratory today. For instance, the analysis of one CSF specimen using current manual methods takes approximately 30-45 minutes. Currently, no automated method for CSF analysis is available on existing hematology platforms.
Automated cell counters or hematology analyzers, such as the ADVIA 120® analyzer (Bayer Corporation, Tarrytown, N.Y.), are typically designed to enumerate cells from samples of whole blood. The standard reagents used on such instruments are designed to compensate for, or take advantage of, various chemical effects of some of the major components of blood plasma, e.g., albumin, lipoprotein, and the like (see, for example, U.S. Pat. Nos. 3,741,875 and 4,412,004). Also, automated analyzers/cell counters typically count from about 2,000 to 50,000 cells in a single blood-dilution per analytical cycle. In addition, the presence of as few as 5 to 10 non-cellular particles (or 5 to 10 cells carried over from a previous cycle, i.e., carryover) which overlap the regions occupied by countable cells, causes only a very small loss of accuracy and/or precision in whole blood sample analysis. Further, instruments such as the ADVIA 120® are typically designed to accept a fixed-sized aliquot of whole blood which is automatically diluted to provide the required cell concentration for a fixed counting period.
Body fluids other than whole blood, for example, cerebrospinal fluid, normally contain no red cells or platelets, little or no dissolved protein and as little as 0.01% of the typical white blood cell count of whole blood. Therefore, if the same automated instruments that handle whole blood cell analyses are also used to determine the cell counts of such other non-blood body fluids, it is required that reagents and dilutions be designed to compensate for the typically nearly acellular conditions of such samples. Moreover, unlike whole blood cell analysis, if the analytical cycle of the instrument handles a volume of body fluid sample that contains only 5 to 10 authentic cells, the interference of non-cellular particles and carryover, referred to above, constitutes a problem in the analysis of such non-blood body fluids that contain very few cells.
Because of the low concentration of cells in samples of non-blood aliquots of body fluids such as CSF, the dilution of sample must be greatly reduced to provide useful precision for the same fixed counting period. Further, rare samples such as CSF are considered to be in a special category compared with whole blood samples. For example, body fluid samples, e.g., CSF, typically arrive in the laboratory infrequently and/or randomly; it is not usually convenient to interrupt the work-flow of the automated analyzer on whole blood samples to accommodate the analysis of the infrequent or random non-blood samples. As it happens, these types of samples are commonly analyzed immediately as “STAT” samples, but they may also be set aside to accumulate for later analysis and more efficient batch-processing. However, such untreated body fluid samples are usually less stable than are typical anti-coagulated whole blood samples, which can often be analyzed with accuracy even after 24 hours of storage at 2-6° C.
It is therefore desirable to be able to mix such non-whole blood body fluid samples with a reagent that fixes the cells in a state that safely permits their later accurate analysis, even after storage for up to 24 or more hours. In addition, for such special procedures involving non-blood body fluid samples, it is usually necessary to develop control materials, which are stable for at least a few months, and which can be used to confirm system gains, when necessary, so that the accuracy of the counts obtained using body fluid samples is assured. The present invention is designed to overcome and address these problems and needs.
In addition, the present invention offers automated methods and procedures, i.e., semi- and fully-automated methods and procedures, for analyzing body fluid samples, such as CSF, and advantageously provides the skilled practitioner with an efficient, reliable and less time-consuming assay for analyzing non-blood samples.